1. Volume 21, Issue 8, Pages 2304-2312 (November 2017)
iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid β Combination for Alzheimer’s Disease 
Takayuki Kondo, Keiko Imamura, Misato Funayama, Kayoko Tsukita, Michiyo Miyake, Akira Ohta, Knut Woltjen, Masato Nakagawa, Takashi Asada, Tetsuaki Arai, Shinobu Kawakatsu, Yuishin Izumi, Ryuji Kaji, Nobuhisa Iwata, Haruhisa Inoue 
Cell Reports 
Volume 21, Issue 8, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2304-2312DOI: (10.1016/j.celrep.2017.10.109)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Transient NGN2 Expression Converts iPSCs into Cortical Neurons
(A) Schema of the differentiation system for iPSC-derived cortical neurons by using the piggyBac vector coding human NGN2 gene, the compound-screening platform, and the in vitro trials.
(B) A generated FAD1-iPSC line expressed the pluripotency markers TRA1-60 (green) and NANOG (red). Scale bar, 200 μm.
(C) Day 8 FAD1 neurons expressed excitatory cortical neuron markers. Left: MAP2 (green) and SATB2 (red). Right: VGLUT1 (green) and TBR2 (red). Scale bars, 200 μm.
(D) Purity of day 8 neurons.
Data indicate mean ± SD (n = 3 for each clone).
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4. Figure 2
Alzheimer’s Disease Patient Cortical Neurons Showed Aβ Phenotypes, Corrected by Genome Editing
(A) Schema of genome editing for PSEN1 exon11, using the CRISPR-Cas9 system.
(B) Sanger-sequence data of the genome-corrected site in PSEN1 G384A.
(C) Aβ phenotypes of iPSC-derived cortical neurons. Data indicate mean ± SD (n = 3 for each clone; ∗p < 0.05, Dunnett’s test for multiple comparisons to FAD1 PBMC origin).
(D) ELISA quantification of Aβ species, altered by adding BSI-IV (β-secretase inhibitor), JNJ (γ-secretase modulator), or semagacestat (γ-secretase inhibitor). Plots show the results of serial 5-fold dilutions ranging from 1.6 nM to 25 μM of the respective compounds. Data indicate mean ± SD (n = 3 for each concentration).
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5. Figure 3 First-Step Screening of a Pharmaceutical Compound Library
(A) Scatterplot graphs of the first-step screening. Fold changes compared with DMSO control were plotted according to each analyte, including Aβ40 (blue), Aβ42 (red), the Aβ42/40 ratio (black), and surviving cells (gray), by adding hit compounds or positive control compounds. The 3SD variance of DMSO control groups is indicated by the yellow band. Compounds causing less survival than 3SD of DMSO control are indicated by overlapping deep red dots. BSI, β-secretase inhibitor; GSM, γ-secretase modulator.
(B) Points represent the Z’ factor of each 96-well plate in the first-step screening.
(C) Criteria of the first-step screening.
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6. Figure 4 Chemical Structure Clustering Identified Six Lead Compounds
(A) Schema of the screening steps.
(B) Chemical-structure clustering classified 129 compounds, filtered by first-step screening, and 54 known Aβ modifiers into 10 clusters, based on ECFP4 fingerprint similarity. The small table shows the number of compounds in each cluster, or known-Aβ modifiers in each cluster, including BSIs or GSMs. BSI-St, statin-derived BSI; BSI-Aq, aminoquinazoline-based BSI; GSM-Ns, NSAIDS-based GSMs; GSM-Im, GSM with imidazole structure.
(C) The Aβ42 alteration ratio (versus DMSO control) of 27 screen hits is shown in the scatterplot graph according to each cluster. Each compound name of the top two compounds to reduce Aβ42 in each cluster is labeled.
(D) The selected six anti-Aβ compounds decreased Aβ production in a dose-dependent manner. Data indicate mean ± SD (n = 3 for each clone).
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7. Figure 5
Combination of Anti-Aβ Compounds Improved Alzheimer’s Disease Phenotypes
Combination of anti-Aβ compounds (BCroT) decreased Aβ production in a dose-dependent manner. Data indicate mean ± SD (n = 3 for each clone). BCroT, combination of bromocriptine, cromolyn, and topiramate.
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8. Figure 6
Anti-Aβ Cocktail of BCroT Decreased Toxic Aβ in Cortical Neurons from a Variety of Individuals
(A) Left: schema of the in vitro trials of BCroT in human iPSC-derived neurons from multiple individuals. BCroT, combination of bromocriptine, cromolyn, and topiramate. Right: patient information and generated iPSC characters. PBMC, peripheral blood mononuclear cell; FAD, familial Alzheimer’s disease; SAD, sporadic Alzheimer’s disease; HC, healthy control.
(B) BCroT suppressed the production of Aβ40 (left), Aβ42 (middle), and the Aβ42/40 ratio (right), even in FAD and SAD neurons other than FAD1 neurons. Data indicate mean ± SD (n = 3 for each clone) (n = 16 clones for each group; ∗p < 0.05).
Cell Reports  , DOI: ( /j.celrep )
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